Mammlian two-hybrid system

ABSTRACT

The invention provides a method of using a reporter gene that encodes a fluorescent polypeptide to indicate that an interaction has occurred between a bait and a prey protein in a mammalian cell. An advantage of using a fluorescent reporter polypeptide is that an interaction between a bait and prey in a mammalian cell can be readily detected, e.g., within 96 hours. In another method of the invention, a prey plasmid contains an Epstein-Barr virus origin of replication (ori-P). The OriP permits the prey plasmid to replicate episomally and indefinitely without damaging the mammalian cell or integrating into the genomic DNA of the mammalian cell. Since such a plasmid is maintained episomally in a circular form, it can be readily recovered from the mammalian cell.

FIELD OF THE INVENTION

[0001] This invention relates to molecular biology.

BACKGROUND OF THE INVENTION

[0002] One approach for elucidating protein-protein binding in cells isthe yeast-based two-hybrid system (Fields and Song (1989) Nature340:245). That system utilizes chimeric genes and detectsprotein-protein interactions via the activation of reporter-geneexpression. Reporter-gene expression occurs as a result ofreconstitution of a functional transcription factor caused by theassociation of fusion proteins encoded by the chimeric genes. Typically,polynucleotides encoding two-hybrid proteins are constructed andintroduced into a yeast host cell. The first hybrid protein consists ofthe yeast Gal4 DNA-binding domain fused to a polypeptide sequence of aknown protein (often referred to as the “bait”). The second hybridprotein consists of the Gal4 activation domain fused to a polypeptidesequence of a second protein (often referred to as the “prey”). Bindingbetween the two-hybrid proteins reconstitutes the Gal4 DNA-bindingdomain with the Gal4 activation domain, which leads to thetranscriptional activation of a reporter gene (e.g., lacZ or HIS3),which is operably linked to a Gal4 binding site.

SUMMARY OF THE INVENTION

[0003] In a first method, expression of a reporter gene that encodes afluorescent polypeptide is used to indicate that an interaction hasoccurred between a bait and a prey protein. An advantage of using afluorescent reporter polypeptide is that an interaction between a baitand prey in a mammalian cell can be readily detected, e.g., within 96hours. In addition, the use of a fluorescent reporter polypeptide allowsthe identification of a single fluorescing mammalian cell withoutfurther manipulation or damage to the cell. For example, a cell thatfluoresces can be identified under a fluorescent microscope. Todetermine the sequence of the prey protein, total DNA from a fluorescingcell is prepared, and the DNA sequence that encodes the prey amplifiedand sequenced.

[0004] In a second method, a prey plasmid containing an Epstein-Barrvirus origin of replication (ori-P) and a bait plasmid are transfectedinto a mammalian cell that expresses Epstein-Barr virus nuclearantigen-1 (EBNA-1). The oriP allows the prey plasmid to replicateepisomally and indefinitely in the cell. Since the prey plasmid ismaintained episomally in a closed circular form, the prey plasmid can bereadily introduced and recovered from a bacterial host cell.

[0005] In one aspect, the invention features a method for detecting aninteraction between a bait and a prey in a mammalian cell. The methodincludes: (a) providing a mammalian cell containing: (i) a reporter geneencoding a fluorescent polypeptide operably linked to a transcriptionalregulatory sequence containing a DNA binding site for a DNA-bindingdomain, (ii) a bait nucleotide sequence encoding a bait fusion protein,including a DNA-binding domain and the bait; (iii) a prey nucleotidesequence encoding a prey fusion protein including a transcriptionalactivation domain and a prey; (b) incubating the cell for 96 hours orless; e.g., 72, 48, 24, or 16 hours; (c) detecting reporter geneexpression, if present, thereby detecting an interaction between thebait and the prey.

[0006] The method can further include isolating total DNA from a cellexpressing the reporter gene, and amplifying the nucleotide sequencethat encodes the bait or prey. In one embodiment, the reporter gene isintegrated into a chromosome of the cell. In another embodiment, thebait or prey is encoded by a nucleotide sequence from a nucleic acidlibrary. The cell can be any mammalian cell, e.g., a primary, secondaryor an immortalized cell such as a CV-1 cell. The reporter gene can be afluorescent polypeptide such as a green fluorescent protein (GFP) or ablue fluorescent protein (BFP).

[0007] In another aspect, the invention features a method for detectingan interaction between a bait and a prey in a mammalian cell. The methodincludes: (a) providing a mammalian cell containing (i) an Epstein-Barrvirus nuclear antigen-1 (EBNA-1); (ii) a reporter gene operably linkedto a transcriptional regulatory sequence containing a DNA binding sitefor a DNA-binding domain, (iii) a bait nucleotide sequence encoding abait fusion protein, including a DNA-binding domain and the bait (e.g.,a known protein), (iv) a prey nucleotide sequence including an origin ofreplication for the Epstein-Barr virus nuclear antigen-1 (oriP) andencoding a prey fusion protein including a transcriptional activationmoiety and the prey (e.g., an unknown protein); and (b) detectingreporter gene expression, if present, thereby detecting an interactionbetween the bait and the prey. The method can further include (c)isolating DNA from a cell expressing the reporter gene; and (d)recovering the nucleotide sequence including the oriP sequence, whichencodes the prey fusion protein. An example of a suitable reporter geneincludes a reporter gene that encodes a fluorescent protein such as agreen fluorescent protein or a blue fluorescent protein. In someembodiments, the reporter gene is integrated into a chromosome of thecell. The cell can be any mammalian cell that expresses EBNA-1 (ormanipulated to express EBNA-1). An example of a mammalian cell includesa cell derived from a primate or a canine. The cell can be a primary,secondary or an immortalized cell. An example of an immortalized cell isa CV-1 cell. The bait and/or the prey can be encoded by a nucleotidesequence from a nucleic acid library.

[0008] The invention also features a kit for detecting an interactionbetween a bait and prey in a mammalian cell. The kit includes: (a) afirst gene construct which includes a regulatory sequence operablylinked to a nucleotide sequence encoding a DNA-binding domain, andwherein the first gene construct includes a cloning site for inserting anucleotide sequence encoding the bait into the first gene construct suchthat the bait is expressed in frame with the DNA-binding domain; (b) asecond gene construct which includes: an oriP sequence, a regulatorysequence operably linked to a nucleotide sequence encoding atranscriptional activation domain, and wherein the second gene constructincludes a cloning site for inserting a nucleotide sequence encoding theprey into the second gene construct such that the prey is expressed inframe with the transcriptional activation domain; (c) a mammalian cellthat expresses an EBNA-1, including a reporter gene encoding afluorescent polypeptide operably linked to a transcriptional regulatorysequence including a DNA binding site for the DNA-binding domain,wherein the reporter gene expresses the fluorescent polypeptide when thebait and prey interact; and (d) instructions for use.

[0009] Also within the scope of the invention is a method of identifyingan agent that disrupts interaction between a bait and a prey, including:(a) providing a mammalian call having: (i) a reporter gene encoding afluorescent polypeptide operably linked to a transcriptional regulatorysequence including a DNA binding site for a DNA-binding domain, (ii) afirst nucleotide sequence encoding a bait fusion protein, including aDNA-binding domain and the bait, (iii) a second nucleotide sequenceencoding a prey fusion protein including a transcriptional activationdomain and the prey; (b) contacting the mammalian cell with a testagent; (c) incubating the cell for 96 hours or less, e.g., 72, 48, 24 or16 hours; and (d) detecting a decrease in expression of the reportergene compared to the level of expression of the reporter gene in amammalian control cell, if present, thereby detecting an agent thatdisrupts interaction between the bait and the prey.

[0010] The invention further features a method of identifying an agentthat enhances interaction between a bait and a prey, including: (a)providing a mammalian cell having: (i) a reporter gene encoding afluorescent polypeptide operably linked to a transcriptional regulatorysequence including a DNA binding site for a DNA-binding domain, (ii) abait nucleotide sequence encoding a bait fusion protein including aDNA-binding domain and the bait, (iii) a prey nucleotide sequenceencoding a prey fusion protein including a transcriptional activationdomain and a prey; (b) incubating the cell for a period of time, e.g.,96 hours or less e.g., 78, 48, 24, or 16 hours; and (c) detecting anincrease in expression of the reporter gene compared to the level ofexpression of the reporter gene in a mammalian control cell, therebydetecting an agent that disrupts interaction between the bait and theprey.

[0011] As used herein, “DNA-binding domain” means an amino acid sequencethat binds specifically to a particular DNA sequence. The site where theDNA-binding domain binds is known as a DNA binding site.

[0012] As used herein, “transcriptional activation domain” means anamino acid sequence which when in proximity to transcriptionalregulatory DNA elements of a target gene, activates gene transcription.

[0013] As used herein, a “reporter gene” means a gene whose expressioncan be assayed.

[0014] As used herein, “interactor” means a protein which is able toform a complex with another protein.

[0015] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. In case of conflict,the present application, including definitions will control. Allpublications, patent applications, patents and other referencesmentioned herein are incorporated by reference.

[0016] Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of theinvention, preferred methods and materials are described below. Thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a schematic drawing of the GAL4-dependent GFP reporterplasmid, pG5GFP.Hyg.

[0018]FIG. 2 is a schematic drawing of a bait expression plasmid,pM.Neo. Also depicted are the unique restriction site(s), EcoR1 andHindIII, for inserting a bait nucleotide sequence, the GAL4 DNA-bindingdomain (GAL4BD), SV40 early promoter (SV40e), and the selection markerneomycin (Neo).

[0019]FIG. 3 is a schematic drawing of a bait expression plasmid,pCMV-DB. Also depicted are the unique restriction site(s), EcoR1 andXbaI, for inserting a bait nucleotide sequence, the GAL4 DNA-bindingdomain (GAL4BD), a CMV promoter, and the selection marker neomycin(Neo).

[0020]FIG. 4 is a schematic drawing of a prey plasmid,pRc/CMV-VP16.OriP. Also depicted are the unique restriction site(s),EcoR1 and XhoI, for inserting a prey nucleotide sequence, the VP16activation domain (VP16), the origin of replication, oriP, and theselection marker ampicillin (Ap).

[0021]FIG. 5 is a schematic drawing of a prey plasmid,pCMV-HA-CR2.ori-P. Also depicted are the unique restriction site(s),EcoR1 and ClaI, for inserting a prey nucleotide sequence, the CR2activation domain (CR2), the origin of replication, oriP, the CMVpromoter and the selection marker ampicillin (Ap).

[0022]FIG. 6 is a schematic drawing of the expression plasmid,pM.CR2.oriP. Also depicted are the GAL4 DNA-binding domain (GAL4BD), theCR2 domain, SV40 early promoter (SV40e), and the selection markerampicillin (Ap).

[0023]FIG. 7 is a schematic drawing depicting a bait-prey interaction.

DETAILED DESCRIPTION

[0024] In one method, a mammalian host cell is engineered such that aninteraction between the bait protein and the prey protein results inexpression of a reporter gene which encodes a fluorescent protein. Thecell in which the prey and bait interact is referred to as a “positive”cell. Cells that fluoresce can be readily detected, e.g., under afluorescent microscope, as early as 16 hours after transfection, and theprey sequence amplified and identified. The advantage of using afluorescent protein as a reporter is that single fluorescent cells canbe readily identified without damaging the cells and without furthermanipulation. For example, the present method overcomes the need foridentifying positive cells based on expression of a drug selectablemarker gene. Drug selection (e.g., G418 or hygromycin B) usuallyrequires too much time for a single screening and the conditions (drugconcentration and selection period) have to be optimized empirically.

[0025] In another method, a prey plasmid containing an Epstein-Barrvirus origin of replication (ori-P) and a bait plasmid are transfectedinto a mammalian cell that expresses Epstein-Barr virus nuclearantigen-1 (EBNA-1). OriP permits the prey plasmid to replicateepisomally and indefinitely without damaging the mammalian cell orintegrating into the genomic DNA of the mammalian cell. Since such aplasmid is maintained episomally in a circular form, it can be readilyintroduced and recovered from a bacterial host. The advantage of usingan oriP prey plasmid is that the use of this plasmid overcomesdegradation problems associated with transient transfection. Normally,transiently introduced plasmids are degraded within several days inmammalian cells if there is no specific mechanism to maintain them. Thisperiod (usually 2 to 3 days) is not long enough to isolate and purifysingle cell-derived colonies expressing the positive prey genes. This isa practical limitation on the efficient isolation of the true positiveclones. The present system overcomes this limitation.

[0026] It will be appreciated by those skilled in the art that manyvariations of the prey and bait fusion proteins can be constructed andis considered within the scope of the present invention. For example, itwill be understood that, for screening polypeptide libraries, thelibrary can be cloned into either the bait or prey fusion proteins. Inthis sense, the terms “prey” and “bait” are merely convenient names forfusion proteins with transcriptional activation domains and DNA-bindingdomains, respectively.

[0027] Each component of the system is now described in more detail.

Bait Protein

[0028] The bait fusion protein includes a fusion between a polypeptidemoiety of interest (e.g., a protein of interest or a polypeptide from apolypeptide library), and a DNA-binding domain which specifically bindsa DNA binding site which occurs upstream of an appropriate reportergene. The nucleotide sequence which encodes the polypeptide moiety ofinterest is cloned in-frame to a nucleotide sequence encoding theDNA-binding domain.

[0029] Any polypeptide that binds a defined DNA sequence can be used asa DNA-binding domain. The DNA-binding domain can be derived from anaturally occurring DNA-binding protein, e.g., a prokaryotic oreukaryotic DNA-binding protein. Alternatively, the DNA-binding domaincan be a polypeptide derived from a protein artificially engineered tointeract with specific DNA sequences. Examples of DNA-binding domainsfrom naturally occurring eukaryotic DNA-binding proteins include p53,Jun, Fos, GCN4, or GAL4. The DNA-binding domain of the bait fusionprotein can also be generated from viral proteins, such as thepappillomavirus E2 protein. In another example, the DNA-binding domainis derived from a prokaryote, e.g., the E. coli LexA repressor can beused, or the DNA-binding domain can be from a bacteriophage, e.g., alambda cI protein. Exemplary prokaryotic DNA-binding domains includeDNA-binding portions of the P22 Arc repressor, MetJ, CENP-B, Rap 1,Xy1S/Ada/AraC, Bir5 and DtxR.

[0030] The DNA-binding protein also can be a non-naturally occurringDNA-binding domain and can be generated by combinatorial mutagenictechniques. Methods for generating novel DNA-binding proteins which canselectively bind to a specific DNA sequence are known in the art. Seee.g., U.S. Pat. No. 5,198,346.

[0031] The basic requirements of the bait fusion protein include theability to specifically bind a defined nucleotide sequence (i.e., a DNAbinding site) upstream of the appropriate reporter gene. The bait fusionprotein should cause little or no transcriptional activation of thereporter gene in the absence of an interacting prey fusion protein. Itis also desirable that the bait not interfere with the ability of theDNA-binding domain to bind to its DNA binding site.

[0032] As appropriate, the DNA-binding domain used in the bait fusionprotein can include oligomerization motifs. It is known in the art thatcertain transcriptional regulators dimerize. Dimerization promotescooperative binding of the transcriptional regulators to their cognateDNA binding sites. For example, where the bait protein includes a LexADNA-binding domain, it can further include a LexA dimerization domain;this optional domain facilitates efficient LexA dimer formation. BecauseLexA binds its DNA binding site as a dimer, inclusion of this domain inthe bait protein also optimizes the efficiency of binding (Golemis andBrent, (1992) Mol. Cell Biol. 12:3006). Other exemplary motifs includethe tetramerization domain of p53 and the tetramerization domain ofBCR-ABL.

[0033] The bait portion of the bait fusion protein may be chosen fromany protein of interest and includes proteins of unknown, known, orsuspected diagnostic, therapeutic, or pharmacological importance. Forexample, the protein of interest can be a protein suspected of being aninhibitor or an activator of a cellular process (e.g., receptorsignaling, apoptosis, cell proliferation, cell differentiation, orimport or export of toxins and nutrients). Examples of bait proteinsinclude oncoproteins such as myc, Ras, Src, Fos; tumor-suppressorproteins such as p53, p21, p16, Rb, and constitutively active Rb withdeleted phosphorylation sites; (Knudsen et. al., Oncogene, 1999,18:5239-45); proteins involved in cell-cycle regulation such as kinasesand phosphates; or proteins involved in signal transduction, e.g.,T-cell signaling, e.g., Zap-70 or SAM-68. The full length of the proteinof interest, or a portion thereof, can be used as the bait protein. Inthe instance when the protein of interest is of a large size, e.g., hasa molecular weight of over 20 kDa, it may be more convenient to use aportion of the protein.

[0034] DNA sequences which encode for the polypeptide of interest andthe DNA-binding domain, e.g., the nucleic acid sequence which encodesfor the GAL4 DNA-binding domain, are inserted into a vector such thatthe desired bait fusion protein is produced in a host mammalian cell.Suitable recombinant expression vectors are known in the art, e.g.,pSG424 (Sadowski and Ptashne, Nucleic Acid Research, 17:7539, 1989), orpM (Clontech, Palo Alto, Calif.). Preferably the recombinant expressionvector includes one or more regulatory sequences operably linked to thefusion nucleic acid sequence to be expressed. The term “regulatorysequence” includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) and these sequences directexpression of the fusion protein.

[0035] Optionally, the vector can also include a selectable marker, theexpression of which in the host mammalian cell permits selection ofcells containing the marker gene from cells that do not contain themarker gene. Selectable markers are known in the art, e.g., neomycin,zeocin or blasticidin.

[0036] Exemplary bait plasmids are shown in FIGS. 3 and 4. To constructthe bait plasmid shown in FIG. 3, pM.Neo, a cDNA of interest can besubcloned into the vector pM (available from Clontech, Palo Alto,Calif.) at the unique EcoR1 and HindIII sites in frame to the N-terminalGAL4 DNA-binding domain (GAL4DB). Alternatively, the pCMV-DB baitplasmid shown in FIG. 4 can be constructed by exising the GAL4DNA-binding domain cDNA from pSG424 (HindIII-EcoR1 fragment) andinserting the fragment into the HindIII/EcoR1 site within the multiplecloning site of the vector pcDNA3 (available from Invitrogen, Carlsbad,Calif.). The bait insert can be cloned into the unique EcoR1/Xba1 site.Expressed mRNA is stabilized by the bovine growth hormone (BGH)polyadenylation sequence.

[0037] Preferably, the vector is integrated into a chromosome of thecell.

[0038] It may also be preferable to introduce an unstructuredpolypeptide linker region between the DNA-binding domain of the fusionprotein and the bait polypeptide sequence. The linker can facilitate,e.g., enhanced flexibility of the fusion protein allowing theDNA-binding domain to freely interact with the DNA binding site.

Prey Fusion Protein

[0039] The prey fusion protein includes a transcriptional activationdomain and a candidate interactor polypeptide sequence which is to betested for its ability to form an intermolecular association with thebait polypeptide. As discussed above, protein-protein contact betweenthe bait and prey fusion proteins (via the interaction of the bait andprey polypeptide portions of these proteins) links the DNA-bindingdomain of the bait fusion protein with the activation domain of the preyfusion protein, generating a protein complex capable of directlyactivating expression of the reporter gene (see FIG. 7).

[0040] Any of a number of activation domains can be used in the preyfusion protein. The activation domain can be a naturally occurringactivation domain, e.g., an activation domain that is derived from aeukaryotic or prokaryotic source. Exemplary activation domains includeGAL4, VP16, CR2, B112, or B17. The activation domain can also be derivedfrom a virus, e.g., VP16 activation domain is derived from herpesvirus.

[0041] DNA sequences which encode the prey and the transcriptionalactivation domain, e.g., a VP16 activation domain, can also includeother sequences such as a nuclear localization sequence (e.g., thosederived from GAL4 or MAT.alpha.2.genes). The nuclear localizationsequence optimizes the efficiency with which prey proteins reach thenuclear-localized reporter gene construct.

[0042] The prey polypeptide can be any polypeptide, e.g., the preypolypeptide can be derived from all or a portion of a known protein or amutant thereof, all or a portion of an unknown protein (e.g., encoded bya gene cloned from a cDNA library), or a random polypeptide sequence.

[0043] To isolate DNA sequences encoding novel interacting proteins,members of a DNA expression library (e.g., a cDNA or synthetic DNAlibrary) can be fused in-frame to the transcriptional activation domainto generate a variegated library of prey fusion proteins.

[0044] In an exemplary embodiment, a cDNA library may be constructedfrom an mRNA population and inserted into an expression vector. Such alibrary of choice may be constructed de novo using commerciallyavailable kits (e.g., from Stratagene, La Jolla, Calif.) or using wellestablished preparative procedures (see, for example, Current Protocolsin Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).Alternatively, a number of cDNA libraries (from a number of differentorganisms) are publicly and commercially available; sources of librariesinclude, e.g., Clontech (Palo Alto, Calif.) and Stratagene (La Jolla,Calif.). It is also noted that prey polypeptides need not be naturallyoccurring full-length proteins. In certain embodiments, prey proteinscan be encoded by synthetic DNA sequences.

[0045] DNA sequences which encode for the prey protein and theactivation domain, e.g., the nucleic acid sequence which encodes for theVP-16 activation domain, are inserted into a vector such that thedesired prey fusion protein is produced in a host mammalian cell. Thevector can be any expression vector as described above. In the instancewhere it is preferable to recover the prey sequence using a bacterialhost cell, as described above, the prey DNA sequences are inserted intoa vector which contains an appropriate origin of replication. By anappropriate origin of replication is meant an origin of replicationwhich allows the vector to be maintained episomally and indefinitelywithout damaging the mammalian host cell or integrating the DNA sequenceinto the genomic DNA of the mammalian host cell. Since the vector ismaintained episomally, the vector can be easily introduced and recoveredfrom a bacterial host cell. An example of such a suitable origin ofreplication is the oriP Epstein-Barr virus replication origin sequence(oriP). In a preferred embodiment, a vector containing an oriP istransformed into a mammalian cell which contains an Epstein Barr virusnuclear antigen-1 (EBNA-1). A vector containing an oriP can replicatestably in a mammalian cell that expresses EBNA-1 (Aiyar et al., EMBOJournal, 17:12:6394-6403).

[0046] Exemplary prey plasmids are shown in FIGS. 4 and 5. The preyplasmid of FIG. 4, pRc/CMV-VP16.OriP, can be constructed by subcloning aprey into the unique EcoRI and XhoI sites of the pCEP4 vector(Invitrogen, Carlsbad, Calif.) to generate fusion proteins with theherpesvirus VP16 transactivator domain harboring an SV40 nuclearlocalization signal sequence. Alternatively, the pCMV-HA-CR2.oriPplasmid can be constructed by removing the EBNA-1 expression cassetteand the hygromycin resistant gene expression cassette from pCEP4(Invitrogen, Carlsbad, Calif.). An expression cassette of a HA-taggedprey can be inserted into the HinDIII/NotI sites within the multiplecloning site of the pCEP4 vector. This plasmid harbours the oriPsequence derived from pCEP4.

Additional Variations of the Prey and Bait Fusion Proteins

[0047] In another aspect of the present invention, the DNA sequenceencoding the prey protein (or alternatively the bait protein) isembedded in a DNA sequence encoding a conformation-constraining protein(i.e., a protein that decreases the flexibility of the amino and carboxytermini of the prey protein). This improves the stability of the fusionprotein structure. Such embodiments are preferred where the preypolypeptide is a relatively short peptide, e.g., 5-25 amino acidresidues. In general, conformation-constraining proteins act asscaffolds or platforms, which limit the number of possiblethree-dimensional configurations the peptide or protein of interest isfree to adopt. Examples of conformation-constraining proteins arethioredoxin or other thioredoxin-like sequences, but many other proteinsare also useful for this purpose.

Reporter Gene

[0048] Expression of the reporter gene indicates an interaction betweenthe prey and bait polypeptides, and permits the identification ofmammalian cells in which an interaction has occurred. The reporter genesequence will include a reporter gene operably linked to a DNA bindingsite to which the DNA-binding domain of the bait fusion protein binds.

[0049] In a preferred embodiment of the invention, the reporter geneencodes a fluorescent molecule, e.g., a green fluorescent protein (GFP)or a blue fluorescent protein (BFP). The advantage of using a reportergene that encodes a fluorescent protein is that a single individualfluorescent positive cell can be identified quickly. For example, usingGFP as the reporter gene product, green fluorescence can be detected asearly as 16 hours after transfection. Positive (fluorescent) cells canbe identified using a fluorescence microscope, .e.g., using an invertedphase-contrast microscope equipped with an epifluorescence light sourceand a fluorescein isothiocyanate filter set. Using this method, positivecells can be identified without damage to the cells, e.g., positive,green fluorescent cells can be easily isolated by conventional cellcloning methods, such as using small plastic cylinders to isolate cells,or collecting positive cells directly using a conventional micropipette(such as Gilson Pipettman). Alternatively, floursesence-activated cellsorter (FACS) can be used to isolate positive cells. However, isolatingpositive cells by FACS is less preferable, since this approach will mixup the positive clones, and hence, may cause cloning bias. The total DNAfrom a positive clone can be prepared by standard procedures and thesequence which encodes the prey protein amplified using PCR andsequenced by standard procedures.

[0050] A preferred fluorescent polypeptide is derived from a GFP. TheGFP gene was originally cloned from the jellyfish Aequorea victoria. Itencodes a protein of 238 amino acids which absorbs blue light (majorpeak at 395 nm) and emits green light (major peak at 509 nm) (Prasher etal., Gene 15:229-223, 1992). GPF genes and functional proteins have beenidentified in a variety of organisms in the phyla hydrozoa, cnidaria,anthozoa and ctenophora. Both wild-type GFP and mutated GFP fromAequorea victoria can be used as a reporter gene. The mutation of GFP(e.g., the substitution of certain amino acids in the GFP polypeptide)has been reported to yield GFP proteins with improved spectralproperties. For example, mutating serine 65 to a threonine generates aGFP variant which has about sixfold greater brightness than wild-typeGFP (Heim et al., Nature 372:663-664, 1995). The coding sequence for anenhanced GFP can be purchased commercially (Clontech, Palo Alto,Calif.). In some embodiments a mammalian-optimized version of a GFP cDNAis used.

[0051] BPF can also be used as a reporter gene. To obtain BFP, tyrosine66 of GFP is mutated to a histidine. This mutated GFP protein fluorescesbright blue, in contrast to the green of the wild-type protein. Othervariants of GFP include yellow fluorescent protein (YFP), and cyanfluorescent protein (CFP). Other suitable fluorescent proteins includethose described by Matz et al., 1999, Nature Biotechnology 17:969-973.

[0052] In the second aspect of the invention, any suitable reporter genecan be used. Examples include chloramphenicol acetyl transferase (CAT;Alton and Vapnek (1979), Nature 282:864-869), and other enzyme detectionsystems, such as beta-galactosidase; firefly luciferase (deWet et al.(1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrechtand Silverman (1984), PNAS 1:4154-4158; Baldwin et al. (1984),Biochemistry 23:3663-3667); phycobiliproteins (especiallyphycoerythrin); alkaline phosphates (Toh et al. (1989) Eur. J Biochem.182:231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2:101), secretedalkaline phosphate (Cullen and Malim (1992) Methods in Enzymol.216:362-368) or fluorescent proteins (e.g., GFP). Other examples ofsuitable reporter genes include those which encode proteins conferringdrug/antibiotic resistance to the host mammalian cell.

[0053] The amount of transcription from the reporter gene may bemeasured using any suitable method. Various suitable methods are knownin the art. For example, specific mRNA expression may be detected usingNorthern blots, or specific protein product may be identified by acharacteristic stain or an intrinsic activity.

[0054] In preferred embodiments, the protein encoded by the reporter isdetected by an intrinsic activity associated with that protein. Forinstance, the reporter gene may encode a gene product that, by enzymaticactivity, gives rise to a detection signal based on, fluorescence,colour, or luminescence.

[0055] In other preferred embodiments, the reporter gene provides aselection method such that cells in which the reporter gene is activatedhave a growth advantage. For example the reporter could enhance cellviability, e.g., by relieving a cell nutritional requirement, and/orprovide resistance to a drug. Another class of useful reporter genesencode cell surface proteins for which antibodies or ligands areavailable. Expression of the reporter gene allows cells to be detectedor affinity purified by the presence of the surface protein.

[0056] In particular embodiments, it may desirable to provide two ormore reporter gene constructs which are regulated by interaction of thebait and prey proteins, e.g., GFP and BFP reporter genes. Thesimultaneous expression of the various reporter genes provides a meansfor distinguishing actual interaction of the bait and prey proteinsfrom, e.g., mutations or other spurious events that activate thereporter gene.

[0057] The reporter gene can be maintained in the mammalian cellepisomally or can be integrated into a chromosome of the mammalian cell.Preferably, the reporter gene is integrated into the chromosone of thegene.

[0058] An exemplary reporter construct is the pG5GFP.Hyg shown inFIG. 1. This plasmid can be constructed as follows. The SV40polyadenylation signal (SmaI-MluI fragment from pGFP-C1, Clontech (PaloAlto, Calif.)) can be inserted downstream to the GFP cDNA (at Not1 sitein the multiple cloning site of pEGFP, Clontech (Palo Alto, Calif.)).The CAT gene of pG5CAT reporter plasmid (Clontech, Palo Alto, Calif.) isreplaced by the GFP cDNA with the SV40 polyadenylation signal,generating pG5GFP. A hygromycin resistant gene expression cassette frompCEP4 (Invitrogen, Carlsbad, Calif.) can be inserted into the Sma! Siteof pGFP, generating pG5GFP.hyg.

Host Cells

[0059] Any cultured mammalian cell can be used in the present mammaliantwo-hybrid system, e.g., a primary, secondary, or immortalized cell.Exemplary mammalian cells are those of mouse, hamster, rat, rabbit, dog,cow, and primate including human. They may be of a wide variety oftissue types, including mast cells, endothelial cells, hepatic cells,kidney cells, or other cell types.

[0060] As used herein, the term primary cell means cells isolated from amammal (e.g., from a tissue source), which are grown in culture for thefirst time before subdivision and transfer to a subculture. The termsecondary cell means cells at all subsequent steps in culturing. Thatis, the first time a plated primary cell is removed from the culturesubstrate and replated (passaged), it is referred to as a secondarycell, as are all cells in subsequent passages. Examples of mammalianprimary and secondary cells which can be transfected includefibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelialcells, intestinal epithelial cells), endothelial cells, glial cells,neural cells, formed elements of the blood (e.g., lymphocytes, bonemarrow cells), muscle cells and precursors of these somatic cell types.

[0061] Immortalized cells are cell lines that exhibit an apparentlyunlimited lifespan in culture. Examples of immortalized human cell linesuseful for the present mammlian two-hybrid system include, but are notlimited to, HT1080 cells (ATCC CCL 121), HeLa cells and derivatives ofHeLa cells (ATCC CCL 2, 2.1 and 2.2), MCF-7 breast cancer cells (ATCCBTH 22), K-562 leukemia cells (ATCC CCL 243), KB carcinoma cells (ATCCCCL 17), 2780AD ovarian carcinoma cells (Van der Blick, A.M. et al.,Cancer Res, 48:5927-5932 (1988), Raji cells (ATCC CCL 86), Jurkat cells(ATCC TIB 152), Namalwa cells (ATCC CRL 1432), HL-60 cells (ATCC CCL240), Daudi cells (ATCC CCL 213), RPMI 8226 cells (ATCC CCL 155), U-937cells (ATCC CRL 1593), Bowes Melanoma cells (ATCC CRL 9607), WI-38VA13subline 2R4 cells (ATCC CLL 75.1), and MOLT-4 cells (ATCC CRL 1582), aswell as heterohybridoma cells produced by fusion of human cells andcells of another species. Secondary human fibroblast strains, such asWI-38 (ATCC CCL 75) and MRC-5 (ATCC CCL 171) may be used. In a preferredembodiment, a CV-1 cell is used.

[0062] In one aspect of the invention, the prey plasmid containing anoriP sequence is transfected into a mammalian cell expressing EBNA-1.Methods of manipulating a mammalian cell to express EBNA-1 are wellknown in the art. For example, the EBNA-1 sequence can be cloned into anexpression vector, transfected into a mammalian cell and expressedtherein. In a preferred embodiment, the mammalian cell into which theoriP prey plasmid is transfected is a mammalian cell that allows theori-P prey plasmid to replicate episomally and indefinitely in the cellwithout causing cell death or transformation. Preferably, the cell isnot a rodent cell. Preferably, the cell is derived from a primate or acanine.

[0063] Methods of transfecting the DNA molecules described herein (e.g.,the reporter gene and associated DNA binding sites, or DNA moleculesthat encode EBNA-1, the bait fusion protein, or prey fusion protein)into a mammalian cell can be carried out using procedures known in theart. Examples of transfection methods include calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, biolistic transfer, or electroporation. Suitable methodsfor transfecting host cells in vitro can be found in Sambrook et al.,eds., Molecular Cloning: A Laboratory Manual (2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and otherlaboratory manuals. In a preferred embodiment, cells are transfectedusing LipofectAMINE PLUS reagent (Gibco/BRL, San Fran., Calif.).

Screening Assays

[0064] The present mammalian two-hybrid system can be used to screen foragents which can act as agonists or antagonists of protein-proteininteractions. In a general sense, the assay evaluates the ability of anagent to modulate binding between the bait and prey polypeptides.Exemplary agents include peptides, nucleic acids, carbohydrates, smallorganic molecules, and natural product extract libraries, such asisolated from animals, plants, fungus and/or microbes.

[0065] In an exemplary screening assay of the present invention, themethods described herein can be used to determine if an agent ofinterest can modulate the binding between a bait and a prey fusionprotein, which are known to interact. The ability of the agent tomodulate the interaction can be determined by detecting an increase ordecrease in reporter gene expression, e.g., expression of a fluorescentprotein, e.g., GFP. Where a decrease in reporter gene expression in thepresence of the agent of interest is detected and compared to a controlcell, the agent is predicted to inhibit the interaction between the baitand prey. Alternatively, where the agent causes an increase in reportergene expression in the presence of an agent of interest, as compared toa control cell, the agent is predicted to enhance the interactionbetween the bait and prey polypeptides. In the control cell, expressionof the reporter gene is determined in the absence of an agent ofinterest.

[0066] The present invention can also be used to screen for agents whichare useful for regulating gene expression in vivo. For example, an agentof interest can be tested for its ability to modulate the interactionbetween a DNA-binding domain and its DNA binding site, where theDNA-binding domain is known to interact with the DNA binding site.

Kit

[0067] The invention provides a kit for detecting interaction between aprotein of interest and a sample protein. In an illustrative embodiment,the kit includes at least one construct and a host cell. In a preferredembodiment, the kit includes: (a) a first gene construct which includesa regulatory sequence operably linked to a nucleotide sequence encodinga DNA-binding domain, and a cloning site, (e.g., a convenient cloningsite which contains a unique restriction site(s)) for inserting anucleotide sequence which encodes a bait. The nucleotide sequence whichencodes the bait is expressed in frame with a DNA-binding domain. Thekit further includes: (b) a second gene construct including an oriPsequence, a regulatory sequence operably linked to a nucleotide sequenceencoding a transcriptional activation domain, and a cloning site, (e.g.,a convenient cloning site which contains a unique restriction site(s))for inserting a nucleotide sequence which encodes a prey such that theprey is expressed in frame with the transcriptional activation domain;(c) a reporter gene construct which contains a sequence which encodes afluorescent polypeptide operably linked to a transcriptional regulatorysequence including a DNA binding site for the DNA-binding domain ofconstruct (a); (d) a mammalian cell that expresses EBNA-1 and (e)instructions for use. In a preferred embodiment, the reporter gene isintegrated into the chromosome of the mammalian cell.

[0068] The kit can also include primers, which can be used to amplifythe prey sequence. Optionally, the kit can also include bacterial cellsinto which one can introduce total DNA from a mammalian cell that wasidentified to contain a positive interaction between the bait and prey.

Other Uses for the Methods Described Herein

[0069] The methods described herein can be used for a variety ofdifferent purposes, e.g., for identifying protein-protein interactions,for generating protein linkage maps, for identifying therapeutictargets, and/or for general cloning strategies.

[0070] The methods can also be used to generate antibody equivalents forspecific determinants, e.g., such as single chain antibodies, minibodiesor the like. For example, a target polypeptide (or epitope thereof) forwhich an antibody or antibody equivalent is sought can be displayed oneither the bait or prey fusion protein. A library of potential bindingpartners can be arrayed on the other fusion protein, as appropriate.Interactions between the target polypeptide and members of the libraryof binding partners can be detected according to methods describedherein.

[0071] Alternatively, the methods described herein can be used to mapresidues of a protein involved in a known protein-protein interaction.Thus, for example, various forms of mutagenesis can be utilized togenerate a combinatorial library of either bait or prey polypeptides,and the ability of the corresponding fusion protein to bind its partnerassayed.

[0072] The present methods could be modified such that the methods couldbe used as a single hybrid method. For example, the method can be usedto clone binding domains specific for a given nucleotide sequence, oralternatively to identify the nucleotide sequence specificity for aknown DNA-binding domain.

EXAMPLE 1 Transfection of a Prey Gene Into a Mammalian Cell

[0073] A cell line that showed high efficiency of transient transfectionand which was found to be a useful as a mammalian host cell in themammalian-two-hybrid system is the CV-I/EBNA-1 cell line. TheCV-I/EBNA-1 is a monkey kidney cell line expressing EBNA-1 by stabletransfection of its expression plasmid. This cell line can be purchasedfrom American Type Culture Collection (ATCC). CV-1/EBNA-1 cells showedvery high efficiency of transient transfection. Using LipofectAMINE PLUSreagent (Gibco/BRL, San Fran., Calif.), up to 50% of the cells werepositively transfected as evaluated by transfection of aβ-galactosidaseexpression plasmid followed by enzymatic in situ cell staining.

[0074] The CV-1/EBNA-1 cell line maintained plasmids containing the oriPepisomally with almost 100% efficiency, and the copy number of themaintained plasmids was about 20 per cell. Since the oriP-plasmids weremaintained episomally in a closed circular form, they were introducedinto bacterial hosts. By simply transforming bacteria with totalcellular DNA prepared from transfected mammalian cells, the plasmid waseasily recovered in Ap-resistant bacterial colonies.

EXAMPLE 2 To Identify Positive Clones

[0075] A Green Fluorescence reporter plasmid, pG5GFP.hyg (FIG. 1), whosetranscription was activated by GAL4 DNA-binding domain fusiontranscriptional activators was constructed. This reporter gene expressesgreen fluorescence when its reporter transcription is activated by aninteraction between a bait and a prey. pG5GFP.hyg did not showsignificant background fluorescence in the absence of two-hybridinteractions.

[0076] A CV-1/EBNA-1 cell line that stably harbors the pG5GFP reporterintegrated within the chromosomal DNA (termed the GalBright cell line)was constructed. This cell line showed green fluorescence only when GAL4DNA-binding domain fusion proteins (baits) and transcriptional activatorfusion proteins (prey) interacted within them. It was very easy toidentify the positive clones under the fluorescence microscope, withoutdamaging the cells. The green fluorescence was detected as early as 16hours after transfection.

[0077] The green fluorescence-positive colonies were easily isolated byconventional cell cloning methods using small plastic cylinders.Isolated colonies were subcultured in separate culture dishes and thegreen fluorescence-positive colonies recloned until all cells growing ina culture dish showed fluorescence.

[0078] This protocol allows the isolation of two-hybrid positive clonesdirectly and quickly without bias-prone enrichment steps or lengthy drugselection.

EXAMPLE 3 Mammalian Two-Hybrid System Using GFP

[0079] As a model two-hybrid interaction, the interaction between aSmad4 C-terminal domain fragment [Smad4(C)] and a MSG1 transcriptionalactivator was tested. The interaction of Smad4 with MSG1 has beenpublished (Shioda et al. PNAS, 95:9785-9790, 1998). Bait fusion proteinscontaining Smads of different lengths were assayed for their ability tobind a prey fusion protein containing MSG1 as follows.

[0080] The pG5GFP reporter (FIG. 1) and a GAL4 DNA-binding domain(GAL4DB) fusion MSG1 transactivator (used as a positive control) weretransiently co-transfected by LipofectAMINE into CV-1/EBNA-1 cells.Strong expression of the green fluorescent protein (GFP) was observed.Transfection efficiency of this cell line by lipofection was up to 50%when evaluated by cotransfecting a β-galactosidase expression plasmidfollowed by in situ enzyme assay. There was no background greenfluorescence when GAL4DB-MSG1 lacking the transactivation domain (theCR2 domain) or GAL4DB without fusion were transfected.

[0081] Transfection of a GAL4DB-smad4(C) (amino acids 302-552) bait didnot result in background fluorescence either. As expected,cotransfection of the GAL4DB-Smad4(C) bait and MSG1 (full-lengthcontaining its intrinsic transactivation domain) resulted in stronginduction of GFP expression. Note that the culture medium contained 10%fetal calf serum throughout the experiments.

[0082] When GALDB-smad4(C) was cotransfected with an MSG1 mutant lackingits Smad4-interaction domain (amino acids 30-60), no green fluorescenceinduced. These results demonstrated strong expression of GFP from thepG5GFP reporter only when a GALb 4DB-bait and an activator-containingprey interacted. Without positive two-hybrid interactions, thebackground GFP expression from this reporter was negligible.

EXAMPLE 4 Construction of Stable CV-1/EBNA-1 Cell Line Containing GFPReporter Gene

[0083] CV-1/EBNA-1 cells were stably transfected with pG5GFP.Hyg(FIG. 1) and selected with hygromycin B for 2 weeks. Among about 300hygromycin-resistant clones, a cell line that showed desirablecharacteristics was selected by functional two-hybrid analysis anddesignated “GalBright” cells. GalBright cells grew as rapidly as theparental cells.

EXAMPLE 5 GalBright Cells and the OriP Sequence

[0084] To determine whether GalBright cells maintain plasmids thatcontain the oriP sequence, oriP sequence-containing test cells weretransiently transfected with expression plasmids for GAL4DB-CR2transactivator (FIG. 6). The GLA4DB-CR2 plasmid was constructed asfollows. The oriP sequence was excised from pCEP4 (AccI-AccI fragment,Invitrogen, Carlsbad, Calif.) and inserted into the pM.CR2 plasmid at anNdeI site, generating pM.CR2.oriP plasimd. Details on the pM.CR2 plasmidare published in Shioda et al., PNAS, 95:9785-9790, 1998. Control cellsthat did not contain the oriP sequence were similarly tranfected. At 24hours after transfection, transfected GalBright cells showed stronggreen fluorescence regardless of the presence or absence of the oriPsequence in the expression plasmids. However, at one week aftertransfection, only those cells that had been transfected withoriP-containing expression plasmid still showed strong greenfluorescence. This demonstrated that GalBright cells maintainedoriP-containing plasmids for a long time and formed green fluorescentcolonies that could be isolated readily from culture dishes byconventional cloning methods.

EXAMPLE 6 Mammalian Cell Two-Hybrid Cloning Procedure

[0085] To isolate cDNA clones for proteins that interact with SMAD4(bait) in mammalian cells, a bait expression plasmid is constructed byfusing cDNA for SMAD4 with GAL4 DNA-binding domain.

[0086] Protein expression of the GAL4DBD-SMAD4 is confirmed by transienttransfection of the plasmid into the GalBright cells followed by Westernblotting using anti-GAL4DBD antibodies, or using anti-SMAD4 antibodies.

[0087] Stably transfected GalBright cells containing the bait plasmidare selected using G418 selection. Stable clones that express the baitprotein (GAL4DBD-SMAD4) are isolated by evaluating clones with Westernblotting using anti-GAL4DBD and anti-SMAD4 antibodies. Several clonesare isolated to ensure successful screening.

[0088] The bait-expressing GalBright Cells are then transfected withprey plasmids, which contain cDNAs derived from a mouse embryo library.The prey plasmid contains the oriP sequence and is transfected into aCV-1 cell expressing EBNA-1 using LipofecAMINE Plus.

[0089] The presence of a green fluorescing cell is determined byfluorescence microscopy, using a conventional FITC filter set.Fluorescence, if present, is detected within 96 hours.

[0090] The green fluorescent GalBright cells are recovered directly byusing a micropipette. In another experiment, green fluorescent GalBrightcells are isolated using conventional cloning cylinders with about 0.5cm diameter and trypsinization.

[0091] On occasion, while recovering the green fluorescent positivecells, negative surrounding cells are also recovered. When this occurs,the single cell suspension of recovered cells are inoculated into newculture dishes with low density so that they will form single-cellderived, isolated colonies. The positive clones are stored in fullfreezing medium at −150° C. or liquid nitrogen.

[0092] To identify the sequence that encodes the prey plasmid, the totalDNA from the positive GalBright cells is prepared using standardmethods. At this point two alternative procedures are used to isolatethe interactor protein.

[0093] In the first procedure, bacteria are transformed with totalcellular DNA preparation. Transformed bacterial clones are plated onLB-Ap plates overnight. Bacterial plasmid DNAs are recovered using astandard plasmid miniprep method. Since GalBright cells can harbor morethan one species of the prey library plasmid, 10 or more bacterialcolonies are reovered and plasmid DNA is prepared.

[0094] The recovered plasmid DNA is then reintroduced into thebait-expressing GalBright cells and non-expressing GalBright cells. Trueprey plasmid give green fluorescence only for the bait-expressingGalBright cells by the two-hybrid interaction, but not for thenon-expressing cells. In some cases where the non-expressing cells showgreen fluorescence, the prey is a nonspecific transcriptional activatorthat activates the reporter gene of the Gal Bright cells independent ofthe bait. Prey plasmids are characterized by amplifying the preysequence by PCR, using primers designed on the vector sequences.

[0095] In the second procedure, the total DNA from positive(fluorescent) cells is isolated. The identity of the sequence thatencodes the prey protein is determined by conventional PCR and sequenceanalysis

[0096] Other embodiments are within the following claims.

What is claimed is:
 1. A method for detecting interaction between a baitand a prey, comprising: (a) providing a mammalian cell comprising: (i) areporter gene encoding a fluorescent polypeptide operably linked to atranscriptional regulatory sequence comprising a DNA binding site for aDNA-binding domain, (ii) a first nucleotide sequence encoding a baitfusion protein, comprising a DNA-binding domain and the bait; (iii) asecond nucleotide sequence encoding prey fusion protein comprising atranscriptional activation domain and the prey; (b) incubating the cellfor 96 hours or less; (c) detecting reporter gene expression, ifpresent, thereby detecting interaction between the bait and the prey. 2.The method of claim 1 , further comprising isolating DNA from a cellexpressing the reporter gene, and amplifying the first or secondnucleotide sequence.
 3. The method of claim 1 , wherein the reportergene is integrated into a chromosome of the cell.
 4. The method of claim1 , wherein the cell is an immortalized cell.
 5. The method of claim 1 ,wherein the cell is a CV-1 cell.
 6. The method of claim 1 , wherein thefluorescent polypeptide is a green fluorescent protein (GFP) or a bluefluorescent protein (BFP).
 7. The method of claim 1 , wherein the baitor prey is encoded by a nucleotide sequence from a nucleic acid library.8. A method for detecting interaction between a bait and a prey,comprising: (a) providing a mammalian cell comprising: (i) an oriPEpstein-Barr virus nuclear antigen-1 (EBNA-1); (ii) a reporter geneoperably linked to a transcriptional regulatory sequence comprising aDNA binding site for a DNA-binding domain, (iii) a first nucleotidesequence encoding a bait fusion protein, comprising a DNA-binding domainand the bait; (iv) a second nucleotide sequence comprising an oriPsequence and encoding a prey fusion protein comprising a transcriptionalactivation domain and the prey; (b) detecting reporter gene expression,if present, thereby detecting interaction between the bait and the prey.9. The method of claim 8 , further comprising: (c) isolating DNA from acell expressing the reporter gene; and (d) recovering the nucleotidesequence comprising the oriP sequence and which encodes the prey. 10.The method of claim 8 , wherein the reporter gene is integrated into achromosome of the cell.
 11. The method of claim 8 , wherein the cell isan immortalized cell.
 12. The method of claim 8 , wherein the cell is aCV-1 cell.
 13. The method of claim 8 , wherein the fluorescentpolypeptide is a green fluorescent protein (GFP) or a blue fluorescentprotein (BFP).
 14. The method of claim 8 , wherein the bait or prey isencoded by a nucleotide sequence from a nucleic acid library.
 15. A kitfor detecting interaction between a bait and a prey in a mammalian cell,comprising: (a) a first gene construct comprising: a regulatory sequenceoperably linked to a nucleotide sequence encoding a DNA-binding domain,and a cloning site for inserting a nucleotide sequence encoding the baitinto the first gene construct such that the bait is expressed in framewith the DNA-binding domain; (b) a second gene construct containing anoriP sequence comprising: a regulatory sequence operably linked to anucleotide sequence encoding a transcriptional activation domain, and acloning site for inserting a nucleotide sequence encoding the prey intothe second gene construct such that the prey is expressed in frame withthe transcriptional activation domain; and (c) a mammalian cell thatexpresses an EBNA-1, comprising a reporter gene encoding a fluorescentpolypeptide operably linked to a transcriptional regulatory sequenceincluding a DNA binding site for the DNA-binding domain, wherein thereporter gene expresses the fluorescent polypeptide when the bait andprey interact; and (d) instructions for use.
 16. A method of identifyingan agent that disrupts interaction between a bait and a prey,comprising: (a) providing a mammalian call comprising: (i) a reportergene encoding a fluorescent polypeptide operably linked to atranscriptional regulatory sequence comprising a DNA binding site for aDNA-binding domain, (ii) a first nucleotide sequence encoding a baitfusion protein, comprising a DNA-binding domain and the bait; (iii) asecond nucleotide sequence encoding a prey fusion protein comprising atranscriptional activation moiety and the prey; (b) contacting themammalian cell with a test agent; (c) incubating the cell for 96 hoursor less; (d) detecting a decrease in expression of the reporter genecompared to the level of expression of the reporter gene in a mammaliancontrol cell, if present, thereby detecting an agent that disruptsinteraction between the bait and the prey.
 17. A method of identifyingan agent that enhances interaction between a bait and a prey,comprising: (a) providing a mammalian cell comprising: (i) a reportergene encoding a fluorescent polypeptide operably linked to atranscriptional regulatory sequence comprising a DNA binding site for aDNA-binding domain, (ii) a first nucleotide sequence encoding a baitfusion protein comprising a DNA-binding domain and the bait; (iii) asecond nucleotide sequence encoding a prey fusion protein comprising atranscriptional activation moiety and a prey; (b) incubating the cellfor 96 hours or less; (c) detecting an increase in expression of thereporter gene compared to the level of expression of the reporter genein a mammalian control cell, thereby detecting an agent that disruptsinteraction between the bait and the prey.